Strain selection of pneumococcal surface proteins

ABSTRACT

The present invention relates to vaccine composition(s) comprising at least two PspAs from strains selected from at least one family, the family being defined by PspAs from strains belonging to the family having greater than or equal to 50% homology in aligned sequences of a C-terminal region of an alpha helical region of PspA. Additionally, the families are further comprised of clades, wherein PspAs from strains which belong to a clade exhibit at least 75% sequence homology in aligned sequences of the C-terminal region of the alpha helix of PspA. Vaccine compositions of the present invention preferably comprise a minimum of 4 and a maximum of 6 strains representing a single clade each, and the at least two PspAs are optionally serologically or broadly cross-reactive.

RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/465,746, filed Jun. 6, 1995, now U.S. Pat. No. 5,679,768, which is acontinuation of application Ser. No. 08/048,896, filed Apr. 20, 1993,now abandoned.

Reference is made to U.S. application Ser. No. 08/714,741, filed Sep.16, 1996, which is a continuation-in-part ("CIP") of U.S. Ser. No.08/529,055, filed Sep. 15, 1995, which is a continuation-in-part of:application Ser. No. 08/226,844, filed May 29, 1992; application Ser.No. 08/093,907, filed Jul. 5, 1994; application Ser. No. 07/889,918,filed Jul. 5, 1994 (corresponding to PCT/US93/05191); application Ser.No. 08/482,981, filed Jun. 7, 1995; application Ser. No. 08/458,399,filed Jun. 2, 1995; application Ser. No. 08/446,201, filed May 19, 1995(as a CIP of U.S. Ser. No. 08/246,636); application Ser. No. 08/246,636,filed May 20, 1994 (as a CIP U.S. Ser. No. 08/048,896, filed Apr. 20,1993 as a CIP of U.S. Ser. No. 07/835,698, filed Feb. 12, 1992 as a CIPof U.S. Ser. No. 07/656,773); application Ser. No. 08/319,795, filedOct. 7, 1994 (as a CIP of U.S. Ser. No. 08/246,636); application Ser.No. 08/072,070, filed Jun. 3, 1993; application Ser. No. 07/656,773,filed Feb. 15, 1991 (U.S. Ser. No. 656,773 and 835,698 corresponding toInt'l application WO 92/1448); application Ser. No. 08/246,636, filedMay 20, 1994 (as a CIP of U.S. Ser. No. 08/048,896); and, each of theseapplications, as well as each document or reference cited in theseapplications, is hereby incorporated herein by reference. Documents orreferences are also cited in the following text, either in a ReferenceList before the claims, or in the text itself; and, each of thesedocuments or references is hereby expressly incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to strain selection of PspAs from strains forvaccine compositions, based upon sequence homology and cross-reactivity.PspA strains can be classified according to sequence homology in theC-terminal region of the alpha helical region, and assigned to a clade,and subsequently, each clade is assigned to a family. Vaccinecompositions of the present invention can comprise, at a minimum 2, andno more than 10 PspAs from strains from each clade, in order to developa broadly efficacious pneumococcal vaccine with a limited number ofstrains.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae is an important cause of otitis media,meningitis, bacteremia and pneumonia, and a leading cause of fatalinfections in the elderly and persons with underlying medicalconditions, such as pulmonary disease, liver disease, alcoholism, sicklecell, cerebrospinal fluid leaks, acquired immune deficiency syndrome(AIDS), and patients undergoing immunosuppressive therapy. It is also aleading cause of morbidity in young children. Pneumococcal infectionscause approximately 40,000 deaths in the U.S. yearly. The most severepneumococcal infections involve invasive meningitis and bacteremiainfections, of which there are 3,000 and 50,000 cases annually,respectively.

Despite the use of antibiotics and vaccines, the prevalence ofpneumococcal infections has declined little over the last twenty-fiveyears; the case-fatality rate for bacteremia is reported to be 15-20% inthe general population, 30-40% in the elderly, and 36% in inner-cityAfrican Americans. Less severe forms of pneumococcal disease arepneumonia, of which there are 500,000 cases annually in the U.S., andotitis media in children, of which there are an estimated 7,000,000cases annually in the U.S. caused by pneumococcus. Strains ofdrug-resistant S. pneumoniae are becoming ever more common in the U.S.and worldwide. In some areas, as many as 30% of pneumococcal isolatesare resistant to penicillin. The increase in antimicrobial resistantpneumococcus further emphasizes the need for preventing pneumococcalinfections.

Pneumococcus asymptomatically colonizes the upper respiratory tract ofnormal individuals; disease often results from the spread of organismsfrom the nasopharynx to other tissues during opportunistic events. Theincidence of carriage in humans varies with age and circumstances.Carrier rates in children are typically higher than those of adults.Studies have demonstrated that 38 to 60% of preschool children, 29 to35% of grammar school children and 9 to 25% of junior high schoolchildren are carriers of pneumococcus. Among adults, the rate ofcarriage drops to 6% for those without children at home, and to 18 to29% for those with children at home. It is not surprising that thehigher rate of carriage in children than in adults parallels theincidence of pneumococcal disease in these populations.

An attractive goal for streptococcal vaccination is to reduce carriagein the vaccinated populations and subsequently reduce the incidence ofpneumococcal disease. There is speculation that a reduction inpneumococcal carriage rates by vaccination could reduce the incidence ofthe disease in non-vaccinated individuals as well as vaccinatedindividuals. This "herd immunity" induced by vaccination against upperrespiratory bacterial pathogens has been observed using the Haemophilusinfluenzae type b conjugate vaccines (Takala, A. K., et al., J. Infect.Dis. 1991; 164: 982-986; Takala, A. K., et al., Pediatr. Infect. Dis.J., 1993; 12: 593-599; Ward, J., et al., Vaccines, S. A. Plotkin and E.A. Mortimer, eds., 1994, pp. 337-386; Murphy, T. V., et al., J.Pediatr., 1993; 122; 517-523; and Mohle-Boetani, J. C., et al., Pediatr.Infect. Dis. J., 1993; 12: 589-593).

It is generally accepted that immunity to Streptococcus pneumoniae canbe mediated by specific antibodies against the polysaccharide capsule ofthe pneumococcus. However, neonates and young children fail to makeadequate immune response against most capsular polysaccharide antigensand can have repeated infections involving the same capsular serotype.One approach to immunizing infants against a number of encapsulatedbacteria is to conjugate the capsular polysaccharide antigens to proteinto make them immunogenic. This approach has been successful, forexample, with Haemophilus influenzae b (see U.S. Pat. No. 4,496,538 toGordon and U.S. Pat. No. 4,673,574 to Anderson).

However, there are over ninety known capsular serotypes of S.pneumoniae, of which twenty-three account for about 95% of the disease.For a pneumococcal polysaccharide-protein conjugate to be successful,the capsular types responsible for most pneumococcal infections wouldhave to be made adequately immunogenic. This approach may be difficult,because the twenty-three polysaccharides included in thepresently-available vaccine are not all adequately immunogenic, even inadults.

Protection mediated by anti-capsular polysaccharide antibody responsesare restricted to the polysaccharide type. Different polysaccharidetypes differentially facilitate virulence in humans and other species.Pneumococcal vaccines have been developed by combining 23 differentcapsular polysaccharides that are the prevalent types of humanpneumococcal disease. These 23 polysaccharide types have been used in alicensed pneumococcal vaccine since 1983 (D. S. Fedson and D. M. Musher,Vaccines, S. A. Plotkin and J. E. A. Montimer, eds., 1994, pp. 517-564).The licensed 23-valent polysaccharide vaccine has a reported efficacy ofapproximately 60% in preventing bacteremia by type pneumococci inhealthy adults.

However, the efficacy of the vaccine has been controversial, and attimes, the justification for the recommended use of the vaccinequestioned. It has been speculated that the efficacy of this vaccine isnegatively affected by having to combine 23 different antigens. Having alarge number of antigens combined in a single formulation may negativelyaffect the antibody responses to individual types within this mixturebecause of antigenic competition. The efficacy is also affected by thefact that the 23 serotypes encompass all serological types associatedwith human infections and carriage.

An alternative approach for protecting children, and also the elderly,from pneumococcal infection would be to identify protein antigens thatcould elicit protective immune responses. Such proteins may serve as avaccine by themselves, may be used in conjunction with successfulpolysaccharide-protein conjugates, or as carriers for polysaccharides.

McDaniel et al. (I), J. Exp. Med. 160:386-397, 1984, relates to theproduction of hybridoma antibodies that recognize cell surfacepolypeptide(s) on S. pneumoniae and protection of mice from infectionwith certain strains of encapsulated pneumococci by such antibodies.

This surface protein antigen has been termed "pneumococcal surfaceprotein A", or "PspA" for short.

McDaniel et al. (II), Microbial Pathogenesis 1:519-531, 1986, relates tostudies on the characterization of the PspA. Considerable diversity inthe PspA molecule in different strains was found, as were differences inthe epitopes recognized by different antibodies.

McDaniel et al. (III), J. Exp. Med. 165:381-394, 1987, relates toimmunization of X-linked immunodeficient (XID) mice withnon-encapsulated pneutnococci expressing PspA, but not isogenicpneumococci lacking PspA, protects mice from subsequent fatal infectionwith pneumococci.

McDaniel et al. (IV), Infect. Immun., 59:222-228, 1991, relates toimmunization of mice with a recombinant full length fragment of PspAthat is able to elicit protection against pneumococcal strains ofcapsular types 6A and 3.

Crain et al, Infect.Immun., 56:3293-3299, 1990, relates to a rabbitantiserum that detects PspA in 100% (n=95) of clinical and laboratoryisolates of strains of S. pneumoniae. When reacted with seven monoclonalantibodies to PspA, fifty-seven S. pneumoniae isolates exhibitedthirty-one different patterns of reactivity.

Above cited applications 08/529,055, filed Sep. 15, 1995, 08/470,626,filed Jun. 6, 1995, 08/467,852, filed Jun. 6, 1995, 08/469,434, filedJun. 6, 1995, 08/468,718, filed Jun. 6, 1995, 08/247,491, filed May 23,1994, 08/214,222, filed Mar. 17, 1994 and 08/214,164, filed Mar. 17,1994, 08/246,636, filed May 20, 1994, and 08/319,795, filed Oct. 7,1994, and U.S. Pat. No. 5,476,929, relate to vaccines comprising PspAand fragments thereof, methods for expressing DNA encoding PspA andfragments thereof, DNA encoding PspA and fragments thereof, the aminoacid sequences of PspA and fragments thereof, compositions containingPspA and fragments thereof and methods of using such compositions.

PspA has been identified as a virulence factor and protective antigen.PspA is a cell surface molecule that is found on all clinical isolates,and the expression of PspA is required for the full virulence ofpneumococci in mouse models (McDaniel et al., (III), J. Esp. Med. 165:381-394, 1987). The biological function of PspA has not been welldefined, although a preliminary report suggests that it may inhibitcomplement activation (Alonso DeVelasco, E., et al., MicrobiologicalRev. 1995; 59: 591-603).

The PspA protein type is independent of capsular type. It would seemthat genetic mutation or exchange in the environment has allowed for thedevelopment of a large pool of strains which are highly diverse withrespect to capsule, PspA, and possibly other molecules with variablestructures. Variability of PspA's from different strains also is evidentin their molecular weights, which range from 67 to 99 kD. The observeddifferences are stably inherited and are not the result of proteindegradation.

Immunization with PspA in a lysate of a recombinant λgt11 clone,elicited protection against challenge with several S. pneumoniae strainsrepresenting different capsular and PspA types, as in McDaniel et al.(IV), Infect. Immun. 59:222-228, 1991. Although clones expressing PspAwere constructed according to that paper, the product was insoluble andisolation from cell fragments following lysis was not possible.

Analysis of the nucleotide and amino acid sequences indicate that thePspA molecule comprises three major regions. The first 288 amino acidsat the amino terminal end of the protein are predicted to have a strongalpha helical structure. The adjacent region of 90 amino acids (289 to369 of Rx1 PspA) has a high density of proline residues; based onsimilar regions in other prokaryotic proteins, this region is believedto transverse the bacterial cell wall. The remaining 196 amino acids atthe carboxyl-terminal end of the molecule (370 to 588 of Rx1 PspA) havea repeated amino acid sequence that has been demonstrated to bind tophosphocholine and lipoteichoic acids. Based on this structure, the PspAmolecule is thought to associate with the inner membrane andlipoteichoic acids via the repeated region in the middle of thecarboxyl-terminal end of the protein. The proline region in the middleof the protein is thought to transverse the cell wall placing the alphahelical region on the outer surface of the S. pneumoniae cells. Thismodel is consistent with the demonstration that the alpha helicalregion, which extends from the surface of the cell, contains theprotective epitopes (Yother, J. et al., J. Bacteriol. 1992; 174:601-609; Yother, J. et al., J. Bacteriol. 1994; 176: 2976-2985;McDaniel, L. S. et al., Microbial Pathog. 1994; 17: 323-337; and Ralph,B. A., et al., Ann. N.Y. Acad. Sci. 1994; 730: 361-363).

Serological analysis of PspA using a panel of seven monoclonalantibodies, indicated that, like capsular polysaccharides, the PspAmolecules are highly diverse among pneumococcal strains. Based on theseanalyses, over 30 PspA protein serotypes were defined, and assignmentsof individual strains into group using a classification system, i.e.,families (or serotypes), were based upon reactivity with the panel ofmonoclonal antibodies. Moreover, SDS-PAGE analysis indicated that withina PspA serotype, further heterogeneity existed on the basis of themolecular size. This diversification further supports the assertion thatPspA is a protective antigen in natural infections; the protectivenature of anti-PspA responses has presumably applied selective pressureon pneumococcus to diversify this molecule. However, thisdiversification of the PspA molecule complicates the development of aPspA vaccine, and leads to the possibility that a PspA vaccine wouldhave to contain many PspA strains, possibly making the vaccineimpractical.

In addition to the published literature specifically referred to above,the inventors, in conjunction with co-workers, have published furtherdetails concerning PspA's, as follows:

1. Abstracts of 89th Annual Meeting of the American Society forMicrobiology, p. 125, item D-257, May 1989;

2. Abstracts of 90th Annual Meeting of the American Society forMicrobiology, p. 98, item D-106, May 1990;

3. Abstracts of 3rd International ASM Conference on StreptococcalGenetics, p. 11, item 12, June 1990;

4. Talkington et al, Infect. Immun. 59:1285-1289, 1991;

5. Yother et al (I), J. Bacteriol. 174:601-609, 1992; and

6. Yother et al (II), J. Bacteriol. 174:610-618, 1992. 7. McDaniel et al(V), Microbiol. Pathogenesis, 13:261-268, 1994.

Alternative vaccination strategies are desirable as such providealternative routes to administration or alternative routes to responses.It would be advantageous to provide an immunological composition orvaccination regimen which elicits protection against various diversifiedpneumococcal strains, without having to combine a large number ofpossibly competitive antigens within the same formulation.

OBJECTS AND SUMMARY OF THE INVENTION

It is an object of the present invention to provide a vaccinecomposition comprising at least two PspAs from strains selected from atleast two families, a family being defined by PspAs from strains havinggreater than or equal to 50% homology in aligned sequences of aC-terminal region of an alpha helix of PspA.

Further, it is an object of the invention to provide vaccinecompositions, wherein the families further comprise one or more clades,wherein clades are defined by one or more PspAs from strains, a PspAfrom a strain belonging to the clade having at least 75% homology withanother PspA from a strain within the clade in the aligned sequences ofthe C-terminal region of the alpha helix of PspA.

Additionally, the present invention provides vaccine compositionswherein the C-terminal region comprises an epitope(s) of interest.

The present invention further provides vaccine compositions wherein acentral domain comprising the C-terminal 100 amino acids of thealpha-helical region (192 to 290 of Rx1 PspA) is an epitope(s) capableof eliciting a protective immune response.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a Pustell DNA matrix analysis of homology between the PspAgenes of Rx1 (ATCC 55334, American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852) and EF10197 strains;

FIG. 2 shows a Pustell protein matrix analysis of homology between thePspA proteins of Rx1 (ATCC 55834) and EF10197 strains;

FIG. 3 shows the sequence identities of PspA clade 1 consensus, andcorresponds to the data presented in Table 3;

FIG. 4 shows the sequence identities to PspA clade 2 consensus, andcorresponds to the data presented in Table 4;

FIG. 5 shows the sequence identities to PspA clade 3 consensus, andcorresponds to the data presented in Table 5;

FIG. 6 shows the sequence identities to PspA clade 4 consensus, andcorresponds to the data presented in Table 6;

FIG. 7 shows the sequence of PspA from strain ATCC6303, a representativestrain of clade 5;

FIG. 8 shows the sequence of PspA from strain BG6380 (ATCC 55838,American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852) a representative strain of clade 6;

FIG. 9 shows the competitive inhibition of rabbit polyclonal anti-Rx1 byPA314, recombinant Rx1 (ATCC 55834) containing amino acids 96 to 314;

FIG. 10 shows the inhibition of polyclonal rabbit anti-Rx1 antibodies byPARx1 and PAEF5668 antigens; and

FIG. 11 shows the inhibition of polyclonal rabbit anti-Rx1 antibodies byPARx1 and PABG6380 antigens.

DETAILED DESCRIPTION

It has now been surprisingly found that, despite the assertions of theprior art regarding the apparent diversity of PspA from strains, theprimary sequence of the alpha helix of PspA has two regions of relativeconservation and a region of extensive diversity between PspAs fromstrains. The two regions of relative conservation are comprised of thefirst, N-terminal, 60 amino acids of the alpha helix, and the last,C-terminal, about 100 amino acids of the alpha helix, as shown in FIGS.1 and 2, wherein the C-terminal end of the alpha helix containscross-reactive and protective epitopes that are critical to thedevelopment of a broadly efficacious PspA vaccine. It has been foundthat any conservation in the first, N-terminal, 60 amino acids of thealpha helix is of little consequence in the cross-reactivity of thestrain, and hence, is irrelevant to the development of a PspA vaccine.

A comparison of the amino acid sequences in the C-terminal region of thealpha helix of PspAs from 24 strains of S. pneumoniae, has revealed thatthe PspA strains can be grouped into 6 clades with greater than 75%homology, and these clades could be grouped into 4 families with greaterthan 50% homology.

Accordingly, the present invention provides a method of strain selectionof PspA, based upon the sequence homology of PspAs in the C-terminalregion of the alpha helix.

A clade is defined herein as comprising PspAs which exhibit greater than75% sequence homology in aligned sequences of the C-terminal region ofthe alpha helix, and a family is defined herein as those clades whichexhibit greater than or equal to 50% homology between member PspAsequences in aligned sequences of the C-terminal region of the alphahelix.

Further, it has been found that in addition to sequence homology,members of a clade exhibit cross-reactivity and cross-protection amongone another, which may suggest a causal relationship between sequencehomology and cross-reactivity. PspAs of strains within the same PspAclade exhibit reciprocal cross-protection from immunization andchallenge experiments. It has not been heretofore recognized in theprior art that there may be such a causal relationship; in fact,families of PspA strains were defined solely on the basis of serologicalcross-reactivity, and based upon the prior art definition of families ofPspA strains, it was believed that the extreme diversity of the PspAmolecule would result in a futile attempt at strain selection. Moreover,the PspA typing system (Crain, et al., Infect. Immun. 59: 222-228, 1990)failed to provide relevant groupings of strains, and suggested anenormous diversity.

Hence, the present invention, in contrast to the teachings of the priorart, enables the selection of PspAs from strains in accordance withsequence homology and cross-reactivity, which facilitates thedevelopment of vaccine compositions comprising multiple PspAs.

The present invention contemplates vaccine compositions comprising twoor more, preferably no more than 10, and more preferably a minimum of 4and a maximum of 6 strains of PspA representing a single clade each, anda pharmaceutically acceptable carrier or diluent. In a preferredembodiment, Rx1, (ATCC 55834) a member of clade 2, is the preferredstrain of clade 2 and/or family 1 which is optionally included in thevaccine composition of the present invention.

The aforementioned definition of a family structure to the C-terminalend of the alpha helix region enables the development of a broadlyefficacious pneumococcal vaccine composition with a limited number ofstrains. Combining strains that represent some or all of the familiesfor this cross-reactive and protective region should provide broadprotection against pneumococcal disease. Not all clades may need to berepresented because of cross-reactions between some clades withinfamilies or because of the epidemiology of these strains or clades inthe population to be vaccinated. However, it is well within the scope ofknowledge of the skilled artisan to determine those strains which shouldbe included within a vaccine composition, without the burden of undueexperimentation.

Additionally, the selected PspAs of the present invention furthercomprise an epitope(s) of interest which can elicit a immune response.An epitope of interest is an antigen or immunogen or immunologicallyactive fragment thereof from a pathogen or toxin of veterinary or humaninterest.

The present invention provides an immunogenic, immunological or vaccinecomposition containing pneumococcal strain(s) having an epitope ofinterest, and a pharmaceutically acceptable carrier or diluent. Animmunological composition containing the PspAs having an epitope ofinterest, elicits an immunological response--local or systemic. Theresponse can, but need not be, protective. An immunogenic compositioncontaining the PspAs having an epitope of interest, likewise elicits alocal or systemic immunological response which can, but need not be,protective. A vaccine composition elicits a local or systemic protectiveresponse. Accordingly, the terms "immunological composition" and"immunogenic composition" include a "vaccine composition" (as the twoformer terms can be protective compositions).

The invention therefore also provides a method of inducing animmunological response in a host mammal comprising administering to thehost an immunogenic, immunological or vaccine composition comprisingPspAs having an epitope of interest, and a pharmaceutically acceptablecarrier or diluent.

As to epitopes of interest, one skilled in the art can determine anepitope of immunodominant region of a peptide or polypeptide, and ergo,the coding DNA therefor from the knowledge of the amino acid andcorresponding DNA sequences of the peptide or polypeptide, as well asfrom the nature of particular amino acids (e.g., size, charge, etc.) andthe codon dictionary, without undue experimentation.

A general method for determining which portions of a protein to use inan immunological composition focuses on the size and sequence of theantigen of interest. "In general, large proteins, because they have morepotential determinants are better antigens than small ones. The moreforeign an antigen, that is the less similar to self configurationswhich induce tolerance, the more effective it is in provoking an immuneresponse." Ivan Roitt, Essential Immunology, 1988.

At a minimum, the peptide can be at least 8 or 9 amino acids long. Thisis the minimum length that a peptide needs to be in order to stimulate aCD4+T cell response which recognizes virus infected cells or cancerouscells). A minimum peptide length of 13 to 25 amino acids is useful tostimulate a CD8+T cell response (which recognizes special antigenpresenting cells which have engulfed the pathogen). See Kendrew, supra.However, as these are minimum lengths, these peptides are likely togenerate an immunological response, i.e., an antibody or T cellresponse; but, for a protective response (as from a vaccinecomposition), a longer peptide is preferred.

With respect to the sequence, the DNA sequence encoding the immunogenicpeptide preferably encodes at least regions of the peptide that generatean antibody response or a T cell response. One method to determine T andB cell epitopes involves epitope mapping. The protein of interest "isfragmented into overlapping peptides with proteolytic enzymes. Theindividual peptides are then tested for their ability to bind to anantibody elicited by the native protein or to induce T cell or B cellactivation. This approach has been particularly useful in mapping T-cellepitopes since the T cell recognizes short linear peptides complexedwith MHC molecules. The method is less effective for determining B-cellepitopes" since B cell epitopes are often not linear amino acid sequencebut rather result from the tertiary structure of the folded threedimensional protein. Janis Kuby, Immunology, (1992) pp. 79-80.

In the case of PspA, the location of the major cross-reactive region atthe C-terminal 100 amino acids of the alpha-helical region was carriedout with recombinant peptides of 100 or more amino acids in length(McDaniel et al., Micro. Pathog. 17: 323-337, 1994).

Another method for determining an epitope of interest is to choose theregions of the protein that are hydrophilic. Hydrophilic residues areoften on the surface of the protein and therefore often the regions ofthe protein which are accessible to the antibody. Janis Kuby,Immunology, (1992) P. 81.

Yet another method for determining an epitope of interest is to performan X-ray cyrstallographic analysis of the antigen (full length)-antibodycomplex. Janis Kuby, Immunology, (1992) p. 80.

Still another method for choosing an epitope of interest which cangenerate a T cell response is to identify from the protein sequencepotential HLA anchor binding motifs which are peptide sequences whichare known to be likely to bind to the MHC molecule.

The peptide which is a putative epitope, to generate a T cell response,should be presented in a MHC complex. The peptide preferably containsappropriate anchor motifs for binding to the MHC molecules, and shouldbind with high enough affinity to generate an immune response. Factorswhich can be considered are: the HLA type of the patient (vertebrate,animal or human) expected to be immunized, the sequence of the protein,the presence of appropriate anchor motifs and the occurance of thepeptide sequence in other vital cells.

An immune response is generated, in general, as follows: T cellsrecognize proteins only when the protein has been cleaved into smallerpeptides and is presented in a complex called the "majorhistocompatability complex MHC" located on another cell's surface. Thereare two classes of MHC complexes--class I and class II, and each classis made up of many different alleles. Different patients have differenttypes of MHC complex alleles; they are said to have a `different HLAtype`.

Class I MHC complexes are found on virtually every cell and presentpeptides from proteins produced inside the cell. Thus, Class I MHCcomplexes are useful for killing cells which when infected by viruses orwhich have become cancerous and as the result of expression of anoncogene. T cells which have a protein called CD4 on their surface, bindto the MHC class I cells and secrete lymphokines. The lymphokinesstimulate a response; cells arrive and kill the viral infected cell.

Class II MHC complexes are found only on antigen-presenting cells andare used to present peptides from circulating pathogens which have beenendocytosed by the antigen-presenting cells. T cells which have aprotein called CD8 bind to the MHC class II cells and kill the cell byexocytosis of lytic granules.

Some guidelines in determining whether a protein is an epitope ofinterest which will stimulate a T cell response, include: Peptidelength--the peptide should be at least 8 or 9 amino acids long to fitinto the MHC class I complex and at least 13-25 amino acids long to fitinto a class II MHC complex. This length is a minimum for the peptide tobind to the MHC complex. It is preferred for the peptides to be longerthan these lengths because cells may cut the expressed peptides. Thepeptide should contain an appropriate anchor motif which will enable itto bind to the various class I or class II molecules with high enoughspecificity to generate an immune response (See Bocchia, M. et al,Specific Binding of Leukemia Oncogene Fusion Protein Peptides to HLAClass I Molecules, Blood 85:2680-2684; Englehard, V H, Structure ofpeptides associated with class I and class II MHC molecules Ann. Rev.Immunol. 12:181 (1994)). This can be done, without undueexperimentation, by comparing the sequence of the protein of interestwith published structures of peptides associated with the MHC molecules.Protein epitopes recognized by T cell receptors are peptides generatedby enzymatic degradation of the protein molecule and are presented onthe cell surface in association with class I or class II MHC molecules.

Further, the skilled artisan can ascertain an epitope of interest bycomparing the protein sequence with sequences listed in the protein database. Regions of the protein which share little or no homology arebetter choices for being an epitope of that protein and are thereforeuseful in a vaccine or immunological composition. Regions which sharegreat homology with widely found sequences present in vital cells shouldbe avoided.

Even further, another method is simply to generate or express portionsof a protein of interest, generate monoclonal antibodies to thoseportions of the protein of interest, and then ascertain whether thoseantibodies inhibit growth in vitro of the pathogen from which theprotein was derived. The skilled artisan can use the other guidelinesset forth in this disclosure and in the art for generating or expressingportions of a protein of interest for analysis as to whether antibodiesthereto inhibit growth in vitro. For example, the skilled artisan cangenerate portions of a protein of interest by: selecting 8 to 9 or 13 to25 amino acid length portions of the protein, selecting hydrophylicregions, selecting portions shown to bind from X-ray data of the antigen(full length)-antibody complex, selecting regions which differ insequence from other proteins, selecting potential HLA anchor bindingmotifs, or any combination of these methods or other methods known inthe art.

Epitopes recognized by antibodies are expressed on the surface of aprotein. To determine the regions of a protein most likely to stimulatean antibody response one skilled in the art can preferably perform anepitope map, using the general methods described above, or other mappingmethods known in the art.

As can be seen from the foregoing, without undue experimentation, fromthis disclosure and the knowledge in the art, the skilled artisan canascertain the amino acid and corresponding DNA sequence of an epitope ofinterest for obtaining a T cell, B cell and/or antibody response. Inaddition, reference is made to Gefter et al., U.S. Pat. No. 5,019,384,issued May 28, 1991, and the documents it cites, incorporated herein byreference (Note especially the "Relevant Literature" section of thispatent, and column 13 of this patent which discloses that: "A largenumber of epitopes have been defined for a wide variety of organisms ofinterest. Of particular interest are those epitopes to whichneutralizing antibodies are directed. Disclosures of such epitopes arein many of the references cited in the Relevant Literature section.")

Further, the invention demonstrates that more than one serologicallycomplementary PspA molecule can be in an antigenic, immunological orvaccine composition, so as to elicit better response, e.g., protection,for instance, against a variety of strains of pneumococci; and, theinvention provides a system of selecting PspAs for a multivalentcomposition which includes cross-protection evaluation so as to providea maximally efficacious composition.

The determination of the amount of antigen, e.g., PspA or truncatedportion thereof and optional adjuvant in the inventive compositions andthe preparation of those compositions can be in accordance with standardtechniques well known to those skilled in the pharmaceutical orveterinary arts. In particular, the amount of antigen and adjuvant inthe inventive compositions and the dosages administered are determinedby techniques well known to those skilled in the medical or veterinaryarts taking into consideration such factors as the particular antigen,the adjuvant (if present), the age, sex, weight, species and conditionof the particular patient, and the route of administration. Forinstance, dosages of particular PspA antigens for suitable hosts inwhich an immunological response is desired, can be readily ascertainedby those skilled in the art from this disclosure, as is the amount ofany adjuvant typically administered therewith. Thus, the skilled artisancan readily determine the amount of antigen and optional adjuvant incompositions and to be administered in methods of the invention.Typically, an adjuvant is commonly used as 0.001 to 50 wt % solution inphosphate buffered saline, and the antigen is present on the order ofmicrograms to milligrams, such as about 0.0001 to about 5 wt %,preferably about 0.0001 to about 1 wt %, most preferably about 0.0001 toabout 0.05 wt % (see, e.g., Examples below or in applications citedherein).

Typically, however, the antigen is present in an amount on the order ofmicrograms to milligrams, or, about 0.001 to about 20 wt %, preferablyabout 0.01 to about 10 wt %, and most preferably about 0.05 to about 5wt %.

Of course, for any composition to be administered to an animal or human,including the components thereof, and for any particular method ofadministration, it is preferred to determine therefor: toxicity, such asby determining the lethal dose (LD) and LD₅₀ in a suitable animal modele.g., rodent such as mouse; and, the dosage of the composition(s),concentration of components therein and timing of administering thecomposition(s), which elicit a suitable immunological response, such asby titrations of sera and analysis thereof for antibodies or antigens,e.g., by ELISA and/or RFFIT analysis. Such determinations do not requireundue experimentation from the knowledge of the skilled artisan, thisdisclosure and the documents cited herein. And, the time for sequentialadministrations can be ascertained without undue experimentation.

Examples of compositions of the invention include liquid preparationsfor orifice, e.g., oral, nasal, anal, vaginal, peroral, intragastric,mucosal (e.g., perlingual, alveolar, gingival, olfactory or respiratorymucosa) etc., administration such as suspensions, syrups or elixirs;and, preparations for parenteral, subcutaneous, intradermal,intramuscular or intravenous administration (e.g., injectableadministration), such as sterile suspensions or emulsions. Suchcompositions may be in admixture with a suitable carrier, diluent, orexcipient such as sterile water, physiological saline, glucose or thelike. The compositions can also be lyophilized. The compositions cancontain auxiliary substances such as wetting or emulsifying agents, pHbuffering agents, gelling or viscosity enhancing additives,preservatives, flavoring agents, colors, and the like, depending uponthe route of administration and the preparation desired. Standard texts,such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985,incorporated herein by reference, may be consulted to prepare suitablepreparations, without undue experimentation.

Compositions of the invention, are conveniently provided as liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsionsor viscous compositions which may be buffered to a selected pH. Ifdigestive tract absorption is preferred, compositions of the inventioncan be in the "solid" form of pills, tablets, capsules, caplets and thelike, including "solid" preparations which are time-released or whichhave a liquid filling, e.g., gelatin covered liquid, whereby the gelatinis dissolved in the stomach for delivery to the gut. If nasal orrespiratory (mucosal) administration is desired, compositions may be ina form and dispensed by a squeeze spray dispenser, pump dispenser oraerosol dispenser. Aerosols are usually under pressure by means of ahydrocarbon. Pump dispensers can preferably dispense a metered dose or,a dose having a particular particle size.

Compositions of the invention can contain pharmaceutically acceptableflavors and/or colors for rendering them more appealing, especially ifthey are administered orally. The viscous compositions may be in theform of gels, lotions, ointments, creams and the like and will typicallycontain a sufficient amount of a thickening agent so that the viscosityis from about 2500 to 6500 cps, although more viscous compositions, evenup to 10,000 cps may be employed. Viscous compositions have a viscositypreferably of 2500 to 5000 cps, since above that range they become moredifficult to administer. However, above that range, the compositions canapproach solid or gelatin forms which are then easily administered as aswallowed pill for oral ingestion.

Liquid preparations are normally easier to prepare than gels, otherviscous compositions, and solid compositions. Additionally, liquidcompositions are somewhat more convenient to administer, especially byinjection or orally, to animals, children, particularly small children,and others who may have difficulty swallowing a pill, tablet, capsule orthe like, or in multi-dose situations. Viscous compositions, on theother hand, can be formulated within the appropriate viscosity range toprovide longer contact periods with mucosa, such as the lining of thestomach or nasal mucosa.

Obviously, the choice of suitable carriers and other additives willdepend on the exact route of administration and the nature of theparticular dosage form, e.g., liquid dosage form (e.g., whether thecomposition is to be formulated into a solution, a suspension, gel oranother liquid form), or solid dosage form (e.g., whether thecomposition is to be formulated into a pill, tablet, capsule, caplet,time release form or liquid-filled form).

Solutions, suspensions and gels, normally contain a major amount ofwater (preferably purified water) in addition to the antigen,lipoprotein and optional adjuvant. Minor amounts of other ingredientssuch as pH adjusters (e.g., a base such as NaOH), emulsifiers ordispersing agents, buffering agents, preservatives, wetting agents,jelling agents, (e.g., methylcellulose), colors and/or flavors may alsobe present. The compositions can be isotonic, i.e., it can have the sameosmotic pressure as blood and lacrimal fluid.

The desired isotonicity of the compositions of this invention may beaccomplished using sodium chloride, or other pharmaceutically acceptableagents such as dextrose, boric acid, sodium tartrate, propylene glycolor other inorganic or organic solutes. Sodium chloride is preferredparticularly for buffers containing sodium ions.

Viscosity of the compositions may be maintained at the selected levelusing a pharmaceutically acceptable thickening agent. Methylcellulose ispreferred because it is readily and economically available and is easyto work with. Other suitable thickening agents include, for example,xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer,and the like. The preferred concentration of the thickener will dependupon the agent selected. The important point is to use an amount whichwill achieve the selected viscosity. Viscous compositions are normallyprepared from solutions by the addition of such thickening agents.

A pharmaceutically acceptable preservative can be employed to increasethe shelf-life of the compositions. Benzyl alcohol may be suitable,although a variety of preservatives including, for example, parabens,thimerosal, chlorobutanol, or benzalkonium chloride may also beemployed. A suitable concentration of the preservative will be from0.02% to 2% based on the total weight although there may be appreciablevariation depending upon the agent selected.

Those skilled in the art will recognize that the components of thecompositions must be selected to be chemically inert with respect to thePspA antigen and optional adjuvant. This will present no problem tothose skilled in chemical and pharmaceutical principles, or problems canbe readily avoided by reference to standard texts or by simpleexperiments (not involving undue experimentation), from this disclosureand the documents cited herein.

The immunologically effective compositions of this invention areprepared by mixing the ingredients following generally acceptedprocedures. For example the selected components may be simply mixed in ablender, or other standard device to produce a concentrated mixturewhich may then be adjusted to the final concentration and viscosity bythe addition of water or thickening agent and possibly a buffer tocontrol pH or an additional solute to control tonicity. Gererally the pHmay be from about 3 to 7.5. Compositions can be administered in dosagesand by techniques well known to those skilled in the medical andveterinary arts taking into consideration such factors as the age, sex,weight, and condition of the particular patient or animal, and thecomposition form used for administration (e.g., solid vs. liquid).Dosages for humans or other mammals can be determined without undueexperimentation by the skilled artisan, from this disclosure, thedocuments cited herein, the Examples below (e.g., from the Examplesinvolving mice).

Suitable regimes for initial administration and booster doses or forsequential administrations also are variable, may include an initialadministration followed by subsequent administrations; but nonetheless,may be ascertained by the skilled artisan, from this disclosure, thedocuments cited herein, and the Examples below.

The following Examples are provided for illustration and are not to beconsidered a limitation of the invention.

EXAMPLES Example 1

Identification of Sequence Homologies Between PspAs

Despite the described diversity of PspA strains, the nucleotide andamino acids sequences of the PspA molecule has been evaluated withrespect to whether any region(s) of conservation have been maintainedwhich could be of utility to vaccine development. The comparison of thenucleotide and amino acid sequences from multiple strains of PspArevealed that the primary sequence of the alpha helix has two regions ofrelative conservation, and a region of extensive diversity betweenstrains. The two regions of diversity are comprised of the first,N-terminal, 60 amino acids of the alpha helix, and the last, C-terminal,100 amino acids of the alpha helix, as shown in FIGS. 1 and 2.

FIG. 1 shows the nucleotide sequences of the alpha helix and prolineregions of the pspA genes from Rx1 (ATCC 55834) and EF10197, bothmembers of the same family or clade, as compared to each other forregions of homology. This comparison was made used a Pustell DNA matrixanalysis within the MacVector version 5.0.2 software, using a window of30 nucleotides, a minimum percentage of homology of 70%, a hash value of6, and a jump value of 1. Points or lines in the graph indicate regionsof homology between the two genes that meet the aforementioned criteria.The results demonstrate homology in the portions of the genes encodingthe N-terminal and C-terminal ends of the alpha helix region, as well asthe proline region.

FIG. 2 shows the amino acid sequence comparison of the alpha helix andproline regions of the PspA proteins from Rx1 and EF10197, both membersof the same family or clade, as compared to each other for regions ofhomology. This comparison was made using a Pustell protein matrixanalysis within the MacVector version 5.0.2 software. The analysis wasdone using a window of 8 amino acids, a minimum percentage homology of70%, a hash value of 2, and the pam250 scoring matrix. Points or linesin the graph indicate regions of homology between the two proteins. Theresults demonstrate homology in the N-terminal and the C-terminal endsof the alpha helix region, as well as in the proline region.

The conserved region at the C-terminal end of the alpha helix regioncorrelated with a region demonstrated to contain protective epitopesthat were conserved between two strains.

Expecting that the C-terminal region of the alpha helix region wascritical to vaccine development, the heterogeneity and family structureof amino acid sequences in this region was examined. The comparison ofthe amino acid sequences in this region between 26 strains of PspArevealed that the PspA strains could be grouped into 6 clades withgreater than 75% homology. These clades could be grouped into 4 familieswith greater than 50% homology. This data is shown in Tables 1 to 6, andFIGS. 3 to 8.

                  TABLE 1    ______________________________________    Family/Clade List                                      % AMINO           HOMOLOGY                   ACID HOMO-           WITHIN                     LOGY TO CLADE    FAMILY FAMILY     CLADE   STRAIN  CONSENSUS    ______________________________________    Family 1           >50%       Clade 1 BG9739  96                              DBL6A   98                              L81905  94                              BG8743  87                              AC94    88                              BG6692  96                              BG8838  95                              DBL1    88                      Clade 2 EF10197 89                              RX1     92                              WU2     87                              0922134 99                              DBL5    92                              BG9163  79                              EF6796  91    Family 2           >50%       Clade 3 EF3296  97                              (ATCC                              558535)                              AC122   96                              BG8090  96    Family 3           >50%       Clade 4 EF5668  92                              (ATCC                              55836)                              BG7817  96                              BG7561  89                              BG11703 100                      Clade 5 ATCC6303                                      100    Family 4           >50%       Clade 6 BG6380  100                              (ATCC                              55838)    ______________________________________

                  TABLE 2A    ______________________________________    Homology Between Clades - Matrix of Amino Acid    Similarity Estimates Between Clades    Clade 1    Clade 2  Clade 3 Clade 4                                       Clade 5                                             Clade 6    ______________________________________    Clade 1           >75%    Clade 2           >50%    >75%    Clade 3           <25%    <20%     >75%    Clade 4           <20%    >30%     >30%  >75%    Clade 5           <20%    <20%     >30%  >50%   >75%    Clade 6           <10%    <20%     <10%  <20%   <20%  >75%    ______________________________________

                  TABLE 2B    ______________________________________    AA% sequence identities to PspA Clade Consensus                           % of AA that                                       % AA iden-          Strain Name (Capsular                           differ from the                                       tity to Clade    Clade Type)            Clade Consensus                                       Consensus    ______________________________________    Clade 1          BG9739 (ATCC 55837) (4)                           4           96          DBL6A (6A)       2           98          L81905 (4)       6           94          BG8743 (23)      13          87          AC94 (9)         12          88          BG6692 (33)      4           96          BG8838 (6)       5           95          DBL1 (6B)        12          88    Clade 2          EF10197 (3)      10          89          RX1 (2)          8           92          WU2 (3)          13          87          0922134 (23)     1           99          DBL5 (5)         8           92          BG9163 (6B)      21          79          EF6796 (6A)      9           91    Clade 3          EF3296 (ATCC 55835) (4)                           1           97          AC122 (9)        2           96          BG8090 (19)      4           96    Clade 4          EF5668 (4)       9           92          BG7817 (7)       4           96          BG7561 (15)      12          89          BG11703 (N.D)    0           100    Clade 5          ATCC6303 (3)     0           100    Clade 6          BG6380 (ATCC 55838) (37)                           0           100    ______________________________________     N.D. = not determined

                  TABLE 3    ______________________________________    Sequence identities to PspA Clade 1 Consensus                                      % AA                       # OF AA THAT   IDENTITY                       DIFFER FROM THE                                      TO CLADE    CLADE  STRAIN NAME CLADE CONSENSUS                                      CONSENSUS    ______________________________________    Clade 1           BG9739 (ATCC                       4              96           55837)           DBL6A       2              98           L81905      6              94           BG8743      13             87           AC94        12             88           BG6692      4              96           BG8838      5              95           DBL1        12             88    ______________________________________

                  TABLE 4    ______________________________________    Sequence identities to PspA Clade 2 consensus                                      % AA                       # OF AA THAT   IDENTITY                       DIFFER FROM THE                                      TO CLADE    CLADE  STRAIN NAME CLADE CONSENSUS                                      CONSENSUS    ______________________________________    Clade 2           EF10197     10             89           RX1 (ATCC   8              92           55834)           WU2         13             87           0922134     1              99           DBL5        8              92           BG9163      21             79           EF6796      9              91           RCT123      3              97           RCT129      1              99           RCT135      0              100           LXS200      0              100    ______________________________________

                  TABLE 5    ______________________________________    Sequence identities to PspA Clade 3 Consensus                                      % AA                       # OF AA THAT   IDENTITY                       DIFFER FROM THE                                      TO CLADE    CLADE  STRAIN NAME CLADE CONSENSUS                                      CONSENSUS    ______________________________________    Clade 3           EF3296 (ATCC                       1              97           55835)           AC122       2              96           BG8090      4              96    ______________________________________

                  TABLE 6    ______________________________________    Sequence identities to PspA Clade 4 Consensus                                      % AA                       # OF AA THAT   IDENTITY                       DIFFER FROM THE                                      TO CLADE    CLADE  STRAIN NAME CLADE CONSENSUS                                      CONSENSUS    ______________________________________    Clade 4           EF5668 (ATCC                       9              92           55836)           BG7817      4              96           BG7561      12             89           BG11703     0              100    ______________________________________

The immunological relevance of these families was demonstrated byserological analysis of S. pneumoniae strains with a large number ofmonoclonal antibodies made to several different PspAs. As shown in Table7, the pattern of reactions with strains in clades 3, 4, 5 and 6 ofmonoclonal antibodies generally correlated with the defined clade bysequence.

                                      TABLE 7    __________________________________________________________________________    Ab Reactions Clades 3-6                Anti-PspA Monoclonal Antibodies                Made to EF3296 (P32)    Made to EF5668 (P56)    STRAIN CLADE*                263D12                    263F6                        264A4                            264A11                                265E6                                    270B6                                        263B7                                            351G12                                                350B4                                                    348G7                                                        350H12    __________________________________________________________________________    EF3296 3    X   X   X   X   X   X   X    (ATCC 55835)    BG7140      X       X   X   X   X    PMsv1281    X   X   X   X       X   X    VH1193      X   X   X   X   X   X   X    EF5668 4                                X   X   X   X    (ATTC 55836)    BG7817 4                                X   X   X   X    BG7561 4                                X   X   X   X    BG11703           4                                X   X   X   X    BG7736                                  X   X   X   X    BG7813                                  X   X   X   X    BG7915                                  X   X   X   X    BG10717/30                              X   X   X   X    ATCC 6306           5                                X   X    BG7619                                  X   X    BG7941                                  X   X    BG13075/30                              X   X    BG6380 6    __________________________________________________________________________     X indicates a positive reaction     *clade was determined by amino acid sequences

Example 2

Competitive Inhibition of Anti-Rx1 Polyclonal Antibodies with the PspAAntigens of Different Strains

Competitive inhibition of anti-PARx1 binding to PARx1 antigen wasanalyzed using a BIAcore® sensory chip, coated with PARx1 antigen. Theresults are shown in FIG. 9. Rabbit polyclonal anti-PARx1 (1200 ng/ml)was allowed to react to the chip either alone, or in the presence ofincreasing concentration of PARx1 antigen (indicated by+in FIG. 9) orPA314 PspA antigen (indicated by squares in FIG. 9); the PA314 PspAantigen contains amino acids 96 to 314 of Rx1(ATCC 55834). Theconcentration of uninhibited antibody able to bind to the PARx1 antigenon the sensory chip surface was measured using mass transportmeasurements on the BIAcore® instrument. The mouse monoclonal IgGanti-PspA antibody, P81-122FI0.A11 was used as a standard for thesemeasurements.

The results of these experiments indicated that the N-terminal conservedregion does not contain antigenic epitopes for the PspA response, andthat the conserved region at the C-terminal end of the alpha helixcontains cross-reactive and protective epitopes that are critical to thedevelopment of a broadly efficacious PspA vaccine. Further, FIG. 9demonstrates the lack of relevance of the first 60 amino acids of theN-terminal region of the alpha helix, as the PA314 PspA antigen used inthe competition assays above, contains amino acids 96 to 314 of Rx1.

FIG. 10 shows the inhibition of PARx1 and PAEF5668 antigens. A BIAcore®sensory chip was coated with PARx1 antigen and rabbit polyclonalanti-PARx1 (7 mM) was allowed to react to the chip either alone, or inthe presence of increasing concentration of PARx1 antigen (representedby squares in FIG. 10) or PAEF5668 antigen (represented by diamonds inFIG. 10). The concentration (mM) of these competitive antigens is shownon the X axis on a logarithmic scale, while the concentration (mM) ofuninhibited polyclonal antibody able to bind to the PARx1 antigen on thesensory chip was measured using mass transport measurements on theBIAcore® instrument, and is shown on the Y axis in FIG. 10.

As expected, the concentration of active, non-competitively inhibitedpolyclonal anti-PARx1 decreased as the concentrations of competitiveinhibitor increased. PARx1 antigen completely inhibited the polyclonalantibodies at sufficient concentrations of antigens in excess. ThePAEF5668 antigen has a maximal inhibition of 8.4%. The mouse monoclonalIgG anti-PspA antibody, P81-122F10.A11 was used as a standard forcalculating the concentrations of active polyclonal antibody in thisassay.

The results of the inhibition study by PARx1 and PABG6380 antigens isshown in FIG. 11. A BIAcore® sensory chip was coated with PARx1 antigenand rabbit polyclonal anti-PARx1 (7 mM) was allowed to react to the chipeither alone, or in the presence of increasing concentration of PARx1antigen (represented by squares in FIG. 11), or PABG6380 antigens(represented by X's in FIG. 11). The concentration (mM) of thesecompetitive antigens is shown on the X axis on a logarithmic scale,while the concentration (mM) of uninhibited polyclonal antibody able tobind to the PARx1 antigen on the sensory chip was measured using masstransport measurements on the BIAcore® instrument, and is shown on the Yaxis in FIG. 11.

As expected, the concentration of active, non-competitively inhibitedpolyclonal anti-PARx1 decreased as the concentration of competitiveinhibitor increased. PARx1 antigen completely inhibited the polyclonalantibodies at sufficient concentrations of antigen in excess. ThePABG6380 antigen did not significantly inhibit the polyclonal antibodyreaction. The mouse monoclonal IgG anti-PspA antibody, P81-122F10.A11was used as a standard for calculating the concentrations of activepolyclonal antibody in the assay.

Further, Table 8 shows the results of inhibition studies of polyclonalrabbit anti-Rx1 antibodies with representative strains of selectiveclades. As shown in the Table, anti-Rx1 antibodies inhibit clade 2effectively, but the inhibition of PspAs in clades which differ from thespecificity of the antibody itself is less effective.

                                      TABLE 8    __________________________________________________________________________    Inhibition of Polyclonal Rabbit anti-Rx1 Antibodies    (Inhibition of Anti-Clade 2 Antibody Reactivity    __________________________________________________________________________    Clade         1    2    2   2   4     6     --    Strain         BG9739              RX1  R36A                       WU2 EF5668                                 BG6380                                       JY1119*         (ATCC              (ATCC        (ATCC (ATCC         55837)              55834)       55836)                                 55638)    Antigen         BG9739/n              PARx1                   R36A/n                       WU2/n                           PAEF5668                                 PABG6380                                       JY1119/n    Name    Antigen         native              recom.**                   native                       native                           recom.**                                 recom.**                                       native    Type    %    35.4 100.0                   100.0                       91.4                           8.4   0.0   0.0    Inhibition    __________________________________________________________________________     *JY1119 is an engineered PspA loss mutant and is used in this assay as a     negative control     **recom. = recombinant

Having thus described in detail certain preferred embodiments of thepresent invention, it is to be understood that the invention defined bythe appended claims is not to be limited by particular details set forthin the above description, as many apparent variations thereof arepossible without departing from the spirit or scope thereof.

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35. Ralph, B. A., D. E. Briles and L. S. McDaniel. 1994. Cross-reactiveprotection eliciting epitopes of pneumoccal surface protein A. Ann N YAcad. Sci. 730: 361-3.

36.. Waltman, W. D., L. S. McDaniel, B. Andersson, L. Bland, B. M. Gray,C. S. Eden and D. E. Briles. 1988. Protein serotyping of Streptococcuspneumoniae based on reactivity to six monoclonal antibodies. Microb.Pathog. 5: 159-67.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 28    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 101 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:1: (xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Lys Glu Ile Asp Glu Ser Asp - # Ser Glu Asp Tyr Val Lys Glu    Gly    #   15    -      Leu Arg Ala Pro Leu Gln Ser Glu - # Leu Asp Asp Ala Lys Gln Ala    Lys    #                 30    -      Leu Ser Lys Leu Glu Glu Leu Ser - # Asp Lys Ile Asp Glu Leu Asp    Ala    #             45    -      Glu Ile Ala Lys Leu Glu Lys Asn - # Val Glu Asp Phe Lys Asn Ser    Asn    #         60    -      Gly Glu Gln Ala Glu Gln Tyr Arg - # Ala Ala Ala Glu Glu Asp Leu    Ala    #     80    -      Ala Lys Gln Ala Glu Leu Glu Lys - # Thr Glu Ala Asp Leu Lys Lys    Ala    #   95    -      Val Asn Glu Pro Glu                     100    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 101 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:2: (xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Lys Glu Ile Asp Glu Ser Asp - # Ser Glu Asp Tyr Val Lys Glu    Gly    #   15    -      Leu Arg Ala Pro Leu Gln Ser Glu - # Leu Asp Asp Ala Lys Gln Ala    Lys    #                 30    -      Leu Ser Lys Leu Glu Glu Leu Ser - # Asp Lys Ile Asp Glu Leu Asp    Ala    #             45    -      Glu Ile Ala Lys Leu Glu Lys Asn - # Val Glu Asp Phe Lys Asn Ser    Asn    #         60    -      Gly Glu Gln Ala Glu Gln Tyr Arg - # Ala Ala Ala Gly Glu Asp Leu    Ala    #     80    -      Ala Lys Gln Ala Glu Leu Glu Lys - # Thr Glu Ala Asp Leu Lys Lys    Ala    #   95    -      Val Asn Glu Pro Glu                     100    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 101 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:3: (xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Lys Glu Ile Asp Glu Ser Asp - # Ser Glu Asp Tyr Val Lys Glu    Gly    #   15    -      Glu Arg Ala Pro Leu Gln Ser Glu - # Leu Asp Asp Ala Lys Gln Ala    Lys    #                 30    -      Leu Ser Lys Leu Glu Glu Leu Ser - # Asp Lys Ile Asp Glu Leu Asp    Ala    #             45    -      Glu Ile Ala Lys Leu Glu Lys Asp - # Val Glu Asp Phe Lys Asn Ser    Asp    #         60    -      Gly Glu Gln Ala Gly Gln Tyr Leu - # Ala Ala Ala Gly Glu Asp Leu    Ile    #     80    -      Ala Lys Lys Ala Glu Leu Glu Lys - # Ala Glu Ala Asp Leu Lys Lys    Ala    #   95    -      Val Asp Glu Pro Glu                     100    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 101 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:4: (xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Lys Glu Ile Asp Glu Ser Asp - # Ser Glu Asp Tyr Val Lys Glu    Gly    #   15    -      Glu Arg Ala Pro Leu Gln Ser Glu - # Leu Asp Asp Ala Lys Gln Ala    Lys    #                 30    -      Leu Ser Lys Leu Glu Glu Leu Ser - # Asp Lys Ile Asp Glu Leu Asp    Ala    #             45    -      Glu Ile Ala Lys Leu Glu Lys Asp - # Val Glu Asp Phe Lys Asn Ser    Asp    #         60    -      Gly Glu Gln Ala Gly Gln Tyr Leu - # Ala Ala Ala Glu Glu Asp Leu    Ile    #     80    -      Ala Lys Lys Ala Glu Leu Glu Gln - # Thr Glu Ala Asp Leu Lys Lys    Ala    #   95    -      Val Asn Glu Pro Glu                     100    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 101 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:5: (xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Lys Glu Ile Asp Glu Ser Asp - # Ser Glu Asp Tyr Val Lys Glu    Gly    #   15    -      Glu Arg Ala Pro Leu Gln Ser Glu - # Leu Asp Asp Ala Lys Gln Ala    Lys    #                 30    -      Leu Ser Lys Leu Glu Glu Xaa Ser - # Asp Lys Xaa Asp Glu Leu Asp    Ala    #             45    -      Glu Ile Ala Lys Leu Glu Lys Asp - # Val Glu Asp Phe Lys Asn Ser    Asp    #         60    -      Gly Glu Gln Ala Gly Gln Tyr Leu - # Ala Ala Ala Glu Glu Asp Leu    Ile    #     80    -      Ala Lys Lys Ala Xaa Leu Glu Lys - # Ala Glu Ala Asp Leu Lys Lys    Ala    #   95    -      Val Asp Glu Pro Glu                     100    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 101 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:6: (xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Lys Glu Ile Asp Glu Ser Asp - # Ser Glu Asp Tyr Ile Lys Glu    Gly    #   15    -      Glu Arg Ala Pro Leu Gln Ser Lys - # Leu Asp Asp Ala Lys Lys Ala    Lys    #                 30    -      Leu Ser Lys Leu Asp Glu Xaa Ser - # Asp Lys Xaa Asp Glu Leu Asp    Ala    #             45    -      Glu Ile Ala Lys Leu Glu Lys Asp - # Val Gly Asp Phe Pro Asn Ser    Asp    #         60    -      Gly Glu Gln Ala Gly Gln Tyr Leu - # Val Ala Ala Glu Lys Asp Leu    Asp    #     80    -      Ala Lys Glu Ala Glu Leu Gly Asn - # Thr Gly Ala Asp Leu Lys Lys    Ala    #   95    -      Val Asp Glu Pro Glu                     100    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 101 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:7: (xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Lys Gly Ile Asp Glu Ser Asp - # Ser Glu Asp Tyr Val Lys Glu    Gly    #   15    -      Leu Arg Ala Pro Leu Gln Ser Glu - # Leu Asp Asp Ala Lys Gln Arg    Thr    #                 30    -      Leu Ser Thr Leu Glu Glu Leu Ser - # Asp Lys Ile Asp Glu Leu Asp    Ala    #             45    -      Glu Ile Ala Lys Leu Glu Lys Asn - # Val Glu Tyr Phe Lys Lys Thr    Asp    #         60    -      Ala Glu Gln Thr Glu Gln Tyr Leu - # Ala Ala Ala Glu Lys Asp Leu    Ala    #     80    -      Asp Lys Lys Ala Glu Leu Glu Lys - # Thr Glu Ala Asp Leu Lys Lys    Ala    #   95    -      Val Asn Glu Pro Glu                     100    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 102 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:8: (xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Lys Glu Ile Asp Glu Ser Asp - # Ser Glu Asp Tyr Val Lys Glu    Gly    #   15    -      Leu Arg Val Pro Leu Gln Ser Glu - # Leu Asp Asp Val Lys Gln Ala    Lys    #                 30    -      Leu Leu Lys Leu Glu Glu Leu Ser - # Asp Lys Ile Asp Glu Leu Asp    Ala    #             45    -      Glu Ile Ala Lys Asn Leu Lys Lys - # Asp Val Glu Asp Phe Gln Asn    Ser    #         60    -      Gly Gly Gly Tyr Ser Ala Leu Tyr - # Leu Glu Ala Ala Glu Lys Asp    Leu    #     80    -      Val Ala Lys Lys Ala Glu Leu Glu - # Lys Thr Glu Ala Asp Leu Lys    Lys    #   95    -      Ala Val Asn Glu Pro Glu                     100    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 99 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:9: (xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Lys Glu Ile Asp Glu Ser Asp - # Ser Glu Asp Tyr Val Lys Glu    Gly    #   15    -      Leu Arg Ala Pro Leu Gln Ser Glu - # Leu Asp Asp Ala Lys Gln Ala    Lys    #                 30    -      Leu Ser Lys Leu Glu Glu Leu Ser - # Asp Lys Ile Asp Glu Leu Asp    Ala    #             45    -      Glu Ile Ala Lys Leu Glu Lys Asp - # Val Glu Asp Phe Lys Asn Ser    Asp    #         60    -      Gly Glu Gln Ala Gln Tyr Leu Ala - # Ala Ala Glu Glu Asp Leu Ala    Lys    #     80    -      Lys Ala Glu Leu Glu Lys Thr Glu - # Ala Asp Leu Lys Lys Ala Val    Asn    #   95    -      Glu Pro Glu    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 99 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:10:(xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Lys Glu Ile Asp Glu Ser Asp - # Ser Glu Asp Tyr Val Lys Glu    Gly    #   15    -      Phe Arg Ala Pro Leu Gln Ser Glu - # Leu Asp Ala Lys Gln Ala Lys    Leu    #                 30    -      Ser Lys Leu Glu Glu Leu Ser Asp - # Lys Ile Asp Glu Leu Asp Ala    Glu    #             45    -      Ile Ala Lys Leu Glu Asp Gln Leu - # Lys Ala Ala Glu Glu Asn Asn    Asn    #         60    -      Val Glu Asp Tyr Phe Lys Glu Gly - # Leu Glu Lys Thr Ile Ala Ala    Lys    #     80    -      Lys Ala Glu Leu Glu Lys Thr Glu - # Ala Asp Leu Lys Lys Ala Val    Asn    #   95    -      Glu Pro Glu    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 99 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:11:(xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Lys Glu Ile Asp Glu Ser Glu - # Ser Glu Asp Tyr Ala Lys Glu    Gly    #   15    -      Phe Arg Ala Pro Leu Gln Ser Lys - # Leu Asp Ala Lys Lys Ala Lys    Leu    #                 30    -      Ser Lys Leu Glu Glu Leu Ser Asp - # Lys Ile Asp Glu Leu Asp Ala    Glu    #             45    -      Ile Ala Lys Leu Glu Asp Gln Leu - # Lys Ala Ala Glu Glu Asn Asn    Asn    #         60    -      Val Glu Asp Tyr Phe Lys Glu Gly - # Leu Glu Lys Thr Ile Ala Ala    Lys    #     80    -      Lys Ala Glu Leu Glu Lys Thr Glu - # Ala Asp Leu Lys Lys Ala Val    Asn    #   95    -      Glu Pro Glu    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 99 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:12:(xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Lys Glu Ile Asp Glu Ser Glu - # Ser Glu Asp Tyr Ala Lys Glu    Gly    #   15    -      Phe Arg Ala Pro Leu His Ser Lys - # Leu Asp Ala Lys Lys Ala Lys    Leu    #                 30    -      Ser Lys Leu Glu Glu Leu Ser Asp - # Lys Ile Asp Glu Leu Asp Ala    Glu    #             45    -      Ile Ala Lys Leu Glu Asp Gln Leu - # Lys Ala Val Glu Glu Asn Asn    Asn    #         60    -      Val Glu Asp Tyr Ser Thr Glu Gly - # Leu Glu Lys Thr Ile Ala Ala    Lys    #     80    -      Lys Thr Glu Leu Glu Lys Thr Glu - # Ala Asp Leu Lys Lys Ala Val    Asn    #   95    -      Glu Pro Glu    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 99 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:13:(xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Lys Asp Ile Asp Glu Ser Asp - # Ser Glu Asp Tyr Ala Lys Glu    Gly    #   15    -      Leu Arg Ala Pro Leu Gln Ser Glu - # Leu Asp Thr Lys Lys Ala Lys    Leu    #                 30    -      Leu Lys Leu Glu Glu Leu Ser Gly - # Lys Ile Glu Glu Leu Asp Ala    Glu    #             45    -      Ile Xaa Glu Leu Glu Val Gln Leu - # Lys Asp Val Glu Gly Asn Asn    Asn    #         60    -      Val Glu Ala Tyr Phe Lys Glu Gly - # Leu Glu Lys Thr Thr Ala Glu    Lys    #     80    -      Ala Thr Glu Leu Glu Lys Ala Glu - # Ala Asp Leu Lys Lys Ala Val    Asp    #   95    -      Glu Pro Glu    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 99 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:14:(xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Glu Glu Ile Asn Glu Ser Asp - # Ser Glu Asp Tyr Ala Lys Glu    Gly    #   15    -      Phe Arg Ala Pro Leu Gln Ser Lys - # Leu Asp Ala Lys Lys Ala Lys    Leu    #                 30    -      Leu Lys Leu Glu Glu Leu Ser Gly - # Lys Ile Glu Glu Leu Asp Ala    Glu    #             45    -      Ile Ala Glu Leu Glu Val Gln Leu - # Lys Asp Val Glu Gly Asn Asn    Asn    #         60    -      Val Glu Ala Tyr Phe Lys Glu Gly - # Leu Glu Lys Thr Thr Ala Glu    Lys    #     80    -      Ala Thr Glu Leu Glu Lys Ala Glu - # Ala Asp Leu Lys Lys Ala Val    Asp    #   95    -      Glu Pro Glu    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 99 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:15:(xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Lys Glu Ile Asp Glu Ser Asp - # Ser Glu Asp Tyr Leu Lys Glu    Gly    #   15    -      Leu Arg Ala Pro Leu Gln Ser Lys - # Leu Asp Thr Lys Lys Ala Lys    Leu    #                 30    -      Ser Lys Leu Glu Glu Leu Ser Asp - # Lys Ile Asp Glu Leu Asp Ala    Glu    #             45    -      Ile Ala Lys Leu Glu Val Gln Leu - # Lys Asp Val Glu Gly Asn Asn    Asn    #         60    -      Val Glu Ala Tyr Phe Lys Glu Gly - # Leu Glu Lys Thr Thr Ala Glu    Lys    #     80    -      Ala Thr Glu Leu Glu Lys Ala Glu - # Ala Asp Leu Lys Lys Ala Val    Asp    #   95    -      Glu Pro Glu    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 99 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:16:(xi) SEQUENCE DESCRIPTION: SEQ    -      Pro Lys Arg Ile Met Ser Leu Ser - # Gln Lys Val Xaa Leu Lys Xaa    Val    #   15    -      Cys Arg Ala Pro Leu Gln Ser Lys - # Leu Asp Ala Gln Lys Ala Glu    Leu    #                 30    -      Leu Lys Leu Glu Glu Leu Ser Gly - # Lys Ile Glu Glu Leu Asp Ala    Glu    #             45    -      Ile Ala Glu Leu Glu Val Gln Leu - # Lys Asp Val Glu Gly Asn Asn    Asn    #         60    -      Val Glu Ala Tyr Phe Lys Glu Gly - # Leu Glu Lys Thr Thr Ala Glu    Lys    #     80    -      Ala Thr Glu Leu Glu Xaa Ala Xaa - # Ala Asp Leu Lys Lys Ala Val    Asp    #   95    -      Glu Pro Glu    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 99 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:17:(xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Lys Glu Ile Asp Glu Ser Asp - # Ser Glu Asp Tyr Ala Lys Glu    Gly    #   15    -      Phe Arg Ala Pro Leu Gln Ser Lys - # Leu Asp Ala Lys Lys Ala Lys    Leu    #                 30    -      Ser Lys Leu Glu Glu Leu Ser Asp - # Lys Ile Asp Glu Leu Asp Ala    Glu    #             45    -      Ile Ala Lys Leu Glu Val Gln Leu - # Lys Asp Ala Glu Gly Asn Asn    Asn    #         60    -      Val Glu Ala Tyr Phe Lys Glu Gly - # Leu Glu Lys Thr Thr Ala Glu    Lys    #     80    -      Ala Thr Glu Leu Glu Lys Ala Glu - # Ala Asp Leu Lys Lys Ala Val    Asp    #   95    -      Glu Pro Glu    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 80 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:18:(xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Asp Lys Glu Ala Gly Glu Ala - # Glu Leu Asp Lys Lys Ala Asp    Gly    #   15    -      Leu Pro Asn Lys Val Ser Asp Leu - # Glu Lys Glu Ile Ser Asn Leu    Glu    #                 30    -      Ile Leu Leu Gly Gly Ala Asp Ser - # Glu Asp Asp Thr Ala Ala Leu    Pro    #             45    -      Asn Lys Leu Ala Thr Lys Lys Ala - # Glu Leu Glu Lys Thr Gln Lys    Glu    #         60    -      Leu Asp Ala Ala Leu Asn Glu Leu - # Gly Pro Asp Gly Asp Glu Glu    Glu    #     80    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 104 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:19:(xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Ala Lys Lys Gln Thr Glu Leu - # Glu Lys Leu Leu Asp Ser Leu    Asp    #   15    -      Pro Glu Gly Lys Thr Gln Asp Glu - # Leu Asp Lys Glu Ala Glu Glu    Ala    #                 30    -      Glu Leu Asp Lys Lys Ala Asp Glu - # Leu Pro Asn Lys Val Ala Asp    Leu    #             45    -      Glu Lys Glu Ile Ser Asn Leu Glu - # Ile Leu Leu Gly Gly Ala Asp    Ser    #         60    -      Glu Asp Asp Thr Ala Ala Leu Pro - # Asn Lys Leu Ala Thr Lys Lys    Ala    #     80    -      Glu Leu Glu Lys Thr Gln Lys Glu - # Leu Asp Ala Ala Leu Asn Glu    Leu    #   95    -      Gly Pro Asp Gly Asp Glu Glu Glu                     100    - (2) INFORMATION FOR SEQ ID NO:20:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 104 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:20:(xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Ala Lys Lys Gln Thr Glu Leu - # Glu Lys Leu Leu Asp Asn Leu    Asp    #   15    -      Pro Glu Gly Lys Thr Gln Asp Glu - # Leu Asp Lys Glu Ala Ala Glu    Ala    #                 30    -      Glu Leu Asp Lys Lys Ala Asp Glu - # Leu Pro Asn Lys Val Ala Asp    Leu    #             45    -      Glu Lys Glu Ile Ser Asn Leu Glu - # Ile Leu Leu Gly Gly Ala Asp    Pro    #         60    -      Glu Asp Asp Thr Ala Ala Leu Pro - # Asn Lys Leu Ala Thr Lys Lys    Ala    #     80    -      Glu Leu Glu Lys Thr Pro Lys Glu - # Leu Asp Ala Ala Leu Asn Glu    Leu    #   95    -      Gly Pro Asp Gly Asp Glu Glu Glu                     100    - (2) INFORMATION FOR SEQ ID NO:21:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 102 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:21:(xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Ala Lys Lys Gln Thr Glu Leu - # Glu Lys Leu Leu Asp Leu Asp    Pro    #   15    -      Glu Gly Lys Thr Gln Asp Glu Leu - # Asp Lys Glu Ala Glu Ala Glu    Leu    #                 30    -      Asp Lys Lys Ala Asp Glu Leu Pro - # Asn Lys Val Ala Asp Leu Glu    Lys    #             45    -      Glu Ile Ser Asn Leu Glu Ile Leu - # Leu Gly Gly Ala Asp Ser Glu    Asp    #         60    -      Asp Thr Ala Ala Leu Pro Asn Lys - # Leu Ala Thr Lys Lys Ala Glu    Leu    #     80    -      Glu Lys Thr Gln Lys Glu Leu Asp - # Ala Ala Leu Asn Glu Leu Gly    Pro    #   95    -      Asp Gly Asp Glu Glu Glu                     100    - (2) INFORMATION FOR SEQ ID NO:22:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 108 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:22:(xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Glu Lys Ala Gly Ala Gly Leu - # Gly Asn Leu Leu Ser Thr Leu    Asp    #   15    -      Pro Glu Gly Lys Thr Gln Asp Glu - # Leu Asp Lys Glu Ala Ala Glu    Ala    #                 30    -      Glu Leu Asn Lys Lys Val Glu Ala - # Leu Pro Asn Gln Val Ser Glu    Leu    #             45    -      Glu Glu Glu Leu Ser Lys Leu Glu - # Asp Asn Leu Lys Asp Ala Glu    Thr    #         60    -      Asn His Val Glu Asp Tyr Ile Lys - # Glu Gly Leu Glu Glu Ala Ile    Ala    #     80    -      Thr Lys Gln Ala Glu Leu Glu Lys - # Thr Pro Lys Glu Leu Asp Ala    Ala    #   95    -      Leu Asn Glu Leu Gly Pro Asp Gly - # Asp Glu Glu Glu    #                105    - (2) INFORMATION FOR SEQ ID NO:23:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 108 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:23:(xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Glu Lys Ala Gly Ala Gly Leu - # Gly Asn Leu Leu Ser Thr Leu    Asp    #   15    -      Pro Glu Gly Lys Thr Gln Asp Glu - # Leu Asp Lys Glu Ala Ala Glu    Ala    #                 30    -      Glu Leu Asn Lys Lys Val Glu Ala - # Leu Pro Asn Gln Val Ser Glu    Leu    #             45    -      Glu Glu Glu Leu Ser Lys Leu Glu - # Asp Asn Leu Lys Asp Ala Glu    Thr    #         60    -      Asn His Val Glu Asp Tyr Ile Lys - # Glu Gly Leu Glu Glu Ala Ile    Ala    #     80    -      Thr Lys Gln Ala Glu Leu Glu Lys - # Thr Pro Lys Glu Leu Asp Ala    Ala    #   95    -      Leu Asn Glu Leu Gly Pro Asp Gly - # Asp Glu Glu Glu    #                105    - (2) INFORMATION FOR SEQ ID NO:24:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 108 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:24:(xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Glu Asp Ala Glu Leu Glu Leu - # Glu Lys Val Leu Ala Thr Leu    Asp    #   15    -      Pro Glu Gly Lys Thr Gln Asp Glu - # Leu Asp Lys Glu Ala Ala Glu    Ala    #                 30    -      Glu Leu Asn Glu Lys Val Glu Ala - # Leu Gln Asn Gln Val Ala Glu    Leu    #             45    -      Glu Glu Glu Leu Ser Lys Leu Glu - # Asp Asn Leu Lys Asp Ala Glu    Thr    #         60    -      Asn Asn Val Glu Asp Tyr Ile Lys - # Glu Gly Leu Glu Glu Ala Ile    Ala    #     80    -      Thr Lys Lys Ala Glu Leu Glu Lys - # Thr Gln Lys Glu Leu Asp Ala    Ala    #   95    -      Leu Asn Glu Leu Gly Pro Asp Gly - # Asp Glu Glu Glu    #                105    - (2) INFORMATION FOR SEQ ID NO:25:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 108 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:25:(xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Glu Lys Ala Glu Ala Glu Leu - # Glu Asn Leu Leu Ser Thr Leu    Asp    #   15    -      Pro Gly Gly Lys Thr Gln Asp Glu - # Leu Asp Lys Gly Ala Ala Glu    Ala    #                 30    -      Glu Leu Asn Lys Lys Val Glu Ala - # Leu Pro Asn Pro Val Xaa Glu    Leu    #             45    -      Glu Glu Glu Leu Ser Pro Pro Glu - # Asp Asn Leu Lys Asp Ala Glu    Thr    #         60    -      Asn His Val Glu Asp Tyr Ile Lys - # Glu Gly Leu Glu Glu Ala Ile    Ala    #     80    -      Thr Lys Gln Ala Glu Leu Glu Glu - # Thr Pro Gln Glu Val Asp Ala    Ala    #   95    -      Leu Asn Asp Leu Val Pro Asp Gly - # Gly Glu Glu Glu    #                105    - (2) INFORMATION FOR SEQ ID NO:26:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 108 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:26:(xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Glu Lys Ala Glu Ala Glu Leu - # Glu Asn Leu Leu Ser Thr Leu    Asp    #   15    -      Pro Glu Gly Lys Thr Gln Asp Glu - # Leu Asp Lys Glu Ala Ala Glu    Ala    #                 30    -      Glu Leu Asn Lys Lys Val Glu Ala - # Leu Pro Asn Gln Val Ser Glu    Leu    #             45    -      Glu Glu Glu Leu Ser Lys Leu Glu - # Asp Asn Leu Lys Asp Ala Glu    Thr    #         60    -      Asn Asn Val Glu Asp Tyr Ile Lys - # Glu Gly Leu Glu Glu Ala Ile    Ala    #     80    -      Thr Lys Gln Ala Glu Leu Glu Lys - # Thr Pro Lys Glu Leu Asp Ala    Ala    #   95    -      Leu Asn Glu Leu Gly Pro Asp Gly - # Asp Glu Glu Glu    #                105    - (2) INFORMATION FOR SEQ ID NO:27:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 119 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:27:(xi) SEQUENCE DESCRIPTION: SEQ    -      Leu Glu Asp Ser Gly Leu Gly Leu - # Glu Lys Val Leu Ala Thr Leu    Asp    #   15    -      Pro Gly Gly Glu Thr Pro Asp Gly - # Leu Asp Lys Glu Ala Ser Glu    Asp    #                 30    -      Ser Asn Ile Gly Ala Leu Pro Asn - # Gln Val Ser Asp Leu Glu Asn    Gln    #             45    -      Val Ser Glu Leu Asp Arg Glu Val - # Thr Arg Leu Pro Ser Asp Leu    Lys    #         60    -      Asp Thr Glu Gly Asn Asn Val Gly - # Asp Tyr Val Lys Gly Gly Leu    Glu    #     80    -      Lys Ala Leu Thr Asp Glu Lys Val - # Gly Leu Asn Asn Thr Pro Lys    Ala    #   95    -      Leu Asp Thr Ala Pro Lys Ala Leu - # Asp Thr Ala Leu Asn Glu Leu    Gly    #                110    -      Pro Asp Gly Asp Glu Glu Glu                 115    - (2) INFORMATION FOR SEQ ID NO:28:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 96 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: Not R - #elevant              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: amino acid    #ID NO:28:(xi) SEQUENCE DESCRIPTION: SEQ    -      Gln Ala Leu Tyr Glu Ser Thr Gln - # Glu Gln Ile Glu Glu Leu Lys    Asp    #   15    -      Tyr Asn Glu Gln Ile Ser Glu Gly - # Glu Glu Thr Leu Ile Leu Ala    Ile    #                 30    -      Gln Asn Lys Ile Ser Asp Leu Asp - # Asp Lys Ile Ala Glu Ala Glu    Lys    #             45    -      Lys Leu Ala Asp Ser Gln Asn Gly - # Glu Gly Val Glu Asp Tyr Trp    Thr    #         60    -      Ser Gly Asp Glu Asp Lys Leu Glu - # Lys Leu Gln Ala Glu Gln Asp    Glu    #     80    -      Leu Gln Ala Glu Leu Asp Gln Leu - # Leu Asp Glu Val Asp Gly Gln    Glu    #   95    __________________________________________________________________________

What we claim is:
 1. A vaccine composition comprising at least twoisolated PspAs from S. pneumoniae strains from at least two PspAfamilies.
 2. A vaccine composition of claim 1, wherein the at least twofamilies further comprise one or more clades, said clades being definedby one or more S. pneumoniae strains.
 3. A vaccine composition of claim2, where the composition further comprises a minimum of 4 isolated PspAsfrom S. pneumoniae strains from said one or more clades.
 4. A vaccinecomposition of claim 2, wherein the composition further comprises amaximum of 6 isolated PspAs from S. pneumoniae strains from said one ormore clades.
 5. A vaccine composition of claim 1, wherein thecomposition further comprises isolated PspA from S. pneumoniae strainRx1 (ATCC 55834).
 6. A vaccine composition of claim 2, wherein thecomposition further comprises isolated PspA from S. pneumoniae strainRx1 (ATCC 55834).
 7. A vaccine composition of claim 3, wherein thecomposition further comprises isolated PspA from S. pneumoniae strainRx1 (ATCC 55834).
 8. A vaccine composition of claim 4, wherein thecomposition further comprises isolated PspA from S. pneumoniae strainRx1 (ATCC 55834).
 9. A vaccine composition comprising at least twoisolated PspAs from S. pneumoniae strains from at least two PspAfamilies having a C-terminal region of an alpha helix of PspA, whereinthe C-terminal region comprises an antigenic epitope of interest whichelicits a protective immunological response against Streptococcuspneumoniae.
 10. A vaccine composition of claim 9, wherein the at leasttwo families further comprise one or more clades, the clades beingdefined by one or more S. pneumoniae strains.
 11. A vaccine compositionof claim 10, wherein the composition further comprises a minimum of 4isolated PspAs from S. pneumoniae strains from the one or more clades.12. A vaccine composition of claim 10, wherein the composition furthercomprises a maximum of 6 isolated PspAs from S. pneumoniae strains fromthe one or more clades.
 13. A vaccine composition of claim 9, whereinthe composition further comprises isolated PspA from S. pneumoniaestrain Rx1 (ATCC 55834).
 14. A vaccine composition of claim 10, whereinthe composition further comprises isolated PspA from S. pneumoniaestrain Rx1 (ATCC 55834).
 15. A vaccine composition of claim 11, whereinthe composition further comprises isolated PspA from S. pneumoniaestrain Rx1 (ATCC 55834).
 16. A vaccine composition of claim 12, whereinthe composition further comprises isolated PspA from S. pneumoniaestrain Rx1 (ATCC 55834).
 17. A vaccine composition of claim 1, whereinthe at least two isolated PspAs are immunologically cross-reactive. 18.A vaccine composition of claim 2, wherein the at least two isolatedPspAs are immunologically cross-reactive.
 19. A vaccine composition ofclaim 9, wherein the at least two isolated PspAs are immunologicallycross-reactive.
 20. A vaccine composition of claim 10, wherein the atleast two isolated PspAs are immunologically cross-reactive.
 21. Avaccine composition comprising at least two isolated PspAs from at leastone PspA family.
 22. A vaccine composition of claim 21, wherein thefamilies further comprise one or more clades, said clades being definedby one or more PspAs.
 23. A vaccine composition of claim 22, wherein thecomposition further comprises a minimum of 4 isolated PspAs from one ormore clades.
 24. A vaccine composition of claim 22, wherein thecomposition further comprises a maximum of 6 isolated PspAs from the oneor more clades.
 25. A vaccine composition of claim 21, wherein thecomposition further comprises isolated PspA from S. pneumoniae strainRx1 (ATCC 55834).
 26. A vaccine composition of claim 22, wherein thecomposition further comprises isolated PspA from S. pneumoniae strainRx1 (ATCC 55834).
 27. A vaccine composition of claim 23, wherein thecomposition further comprises isolated PspA from S. pneumoniae strainRx1 (ATCC 55834).
 28. A vaccine composition of claim 24, wherein thecomposition further comprises isolated PspA from S. pneumoniae strainRx1 (ATCC 55834).
 29. A vaccine composition comprising at least twoisolated PspAs from at least two PspA families, said families having aC-terminal region of an alpha helix of PspA, wherein the C-terminalregion comprises an antigenic epitope of interest which elicits aprotective immunological response against S. pneumoniae.
 30. A vaccinecomposition of claim 29, wherein the at least two families furthercomprise one or more clades, the clades being defined by one or morePspAs.
 31. A vaccine composition of claim 30, wherein the compositionfurther comprises a minimum of 4 isolated PspAs from the one or moreclades.
 32. A vaccine composition of claim 30, wherein the compositionfurther comprises a maximum of 6 isolated PspAs from the one or moreclades.
 33. A vaccine composition of claim 29, wherein the compositionfurther comprises isolated PspA from S. pneumoniae strain Rx1 (ATCC55834).
 34. A vaccine composition of claim 30, wherein the compositionfurther comprises isolated PspA from S. pneumoniae strain Rx1 (ATCC55834).
 35. A vaccine composition of claim 31, wherein the compositionfurther comprises isolated PspA from S. pneumoniae strain Rx1 (ATCC55834).
 36. A vaccine composition of claim 32, wherein the compositionfurther comprises isolated PspA from S. pneumoniae strain Rx1 (ATCC55834).
 37. A vaccine composition of claim 21, wherein the at least twoisolated PspAs are immunologically cross-reactive.
 38. A vaccinecomposition of claim 22, wherein the at least two isolated PspAs areimmunologically cross-reactive.
 39. A vaccine composition of claim 29,wherein the at least two isolated PspAs are immunologicallycross-reactive.
 40. A vaccine composition of claim 30, wherein the atleast two isolated PspAs are immunologically cross-reactive.